基于4300 DNA分析系统的茶树SSR发掘方法优化与建立

Establishment a SSR-PCR System of Tea Plant [Camellia sinensis(L.) O. Kuntze] Based on the 4300 DNA Analysis System

简单序列重复(Simple sequence repeats,SSR)在茶树遗传与育种研究中具有重要的作用,基于4300DNA分析系统的SSR发掘具有通量高、准确和灵敏等特点,已被用于许多物种的分子标记研究,但在茶树的相关研究中尚未见报道。本研究应用单因素试验和L9(34)正交设计试验对影响茶树SSR-PCR的主要参数进行优化,建立了适合于4300 DNA分析系统的茶树SSR-PCR反应体系:1.0μL DNA(25 ng·μL-1),0.2μL M13F-F、0.2μL R和0.4μL IR-M13F,0.8μL dNTPs(25 mmol·L-1),1.0μL 10×Buffer(含Mg2+),0.1μL Ex-Taq聚合酶(5 U·μL-1),无菌水定容至10μL。所有引物浓度均为1μmol·L-1。同时,本研究还证明,可以以自行配制的6.5%聚丙烯酰胺凝胶溶液(acry∶bis=29∶1)替代4300 DNA分析系统指定凝胶溶液,检测SSR位点。

Simple sequence repeats（SSR） play an important role in genetic and breeding research for tea plant [Camellia sinensis（L.） O. Kuntze]. 4300 DNA Analysis System is high-throughput, accurate and sensitive in detecting the signals, it has been used in molecular markers techniques in many species, but its application in tea plant has not been reported up to now. In this study, the main parameters that affect tea SSR-PCR have been optimized and verified via single factor experiment and L9（34） orthogonal test. A suitable SSR-PCR reaction system of the 4300 DNA Analysis System for the tea plant genetic research was obtained： 1.0 μL DNA（25 ng·μL^-1）, 0.2 μL MF-F, 0.2 μL R and 0.40 μL IR-MF, 0.8 μL of dNTPs（25 mmol·L^-1）, 1.0 μL 10×Buffer（Mg^2＋）, 0.1 μL Ex-Taq polymerase（5 U·μL^-1）, adding sterile water to a total volume of 10 μL. Concentration of all primers is 1 μmol·L^-1. Besides, in this research, polyacrylamide gel solution（acry∶bis is 29∶1, concentration is 6.5%） self-made is detected to be alternative to the gel solution provided by 4300 DNA analysis system for detecting SSR loci.